The presence of phonon phenomena induced in DNA by radiation in the 75 GHz to 5 THz frequency range have now been demonstrated to result in resonance absorption properties unique to a particular DNA molecule. The present invention relates to a method and apparatus for measuring the phonon resonance occurring in a DNA molecule when the molecule is subjected to electromagnetic radiation in the 75 GHz through 5 THz frequency range. The method and apparatus of the present invention are useful in locating and identifying DNA molecules of interest and in determining damage to known DNA samples.
The genetic molecule deoxyribonucleic acid (DNA) is constantly exposed to a variety of chemical and physical agents resulting in changes to the structure of this molecule. These changes in the structure of the DNA molecule can interfere with replication and transcription of DNA and are generally referred to as DNA damage. Biological consequences of DNA damage include cell death and mutations, events that may cause cancers, mental retardation and reduced growth and development.
Various methods exist for detecting DNA damage. For example, photodamage in DNA resulting from ultraviolet radiation can be detected by chromatography, enzymatic and biochemical incision of DNA at sites of photoproducts or antibody binding to structural damage in DNA. The cyclobutane dimer was first detected in DNA using two-dimensional paper chromatography. Other types of base damage can be determined via techniques such as thin-layer chromatography and high pressure liquid chromatography. Other procedures measure strand breaks induced directly in DNA via an agent or via enzymatic or biochemical treatments that cleave DNA at damaged sites. For example uvrABC exinuclease, a partial excision repair complex purified from Escherichia coli, cleaves DNA on either side of damage produced by exposure to genotoxic chemicals or ultraviolet radiation. More recently, the ability of endonuclease VII to cleave at mispairings in double-stranded DNA has been exploited for enzymatic mutation detection (Youil et al. Proc. Nat""l Acad. Sci. USA 1995 92:87-9). Further, Golz et al. have disclosed improved reaction conditions which increase the selectivity of endonuclease VII for mismatches up to 500 fold (Mutat. Res. 1998 382(3-4):85-92). Immunochemical approaches adapted to the analysis of DNA damage include immunoassays, immunofluorescence, immunoprecipitation, enzyme-linked radioimmunoassays, and quantitative and immunoelectron microscopy. In addition, an ultrasensitive method for measuring DNA damage was recently described. This method couples immunochemical recognition with capillary electrophoresis and laser-induced fluorescence detection (Le et al. Science 1998 280 30 (5366):1101-2).
However, these methods for detecting DNA damage are indirect and relatively cumbersome. Further, they are not suitable for field detection in a stand-off mode.
Recent advances in understanding the interaction between microwave/millimeter wave radiation and living matter have opened new avenues in the detection and identification of microorganisms. In particular, DNA has been suggested to interact with electromagnetic radiation in the millimeter wave regions of the spectrum, due to the presence of phonon modes and plasmon modes of base pairs along the double helix of the DNA chain (Saxena, V. K. and Van Zandt, L. L. Phys. Rev. A 1989 40:6134; Saxena et al. Phys. Rev. A 1989 39: 1474; Van Zandt, L. L. and Saxena, V. K. Phys. Rev. A 1990 42:4993; Saxena, V. K. and Van Zandt, L. L. Phys. Rev. A 1992 45:7610; and Smith et al. IEEE J. Quantum Elec. 1988 15 24:255).
A modified self-consistent phonon approximation theory has been used to calculate temperature dependent interbase hydrogen bond disruption profiles for a number of six base pair repeating sequence infinite B-DNA polymers with various guanine-cytosine/adenine-thymine ratios (Chen, Y. Z. and Prohofsky, E. W. Eur. Biophys. J. 1996 25(1):9-18). Calculations via this modified phonon approximation theory were used effectively to calculate H-bond disruption behavior of different DNA sequences.
The expected absorption of microwave radiation in the GHz frequency range by fixed-length DNA polymer molecules dissolved in saline solution have also been calculated (Biopolymers 1989 28(8):1429-33).
Further, the feasibility of using spectroscopic techniques, and more specifically microwave and millimeter wave technology, as a probe for possible detection of damage to DNA has been examined by Woolard et al. (J. Of Applied Toxicology 1997 17(4):243-246). In this study, a series of resonances were first predicted, based on available physical parameter values and reasonable assumptions, in a spectral region with a frequency at 88, 89, 110, 172, 232, 300, 382, 418, 503, 561, 638, 784, 891, 920 and 1019 GHz. Preliminary experiments were then conducted to detect some of the resonances using microwave absorption spectroscopy. More specifically, DNA samples from relatively similar species, salmon and herring, were mechanically loaded as dry DNA salts into a 100-mil long shortened section of waveguide and microwave scattering parameter (S11) data was generated via an HP 8510 W-band (i.e. 85-110 GHz) tester. A variance in the occurrence of resonance behavior as a function of electromagnetic energy frequency between the DNA samples of the different species was observed. Microwave absorption spectrum of the same dry sodium herring DNA sample in the frequency region of 180-220 GHz were also measured via a Millitech frequency domain-up conversion unit transmitted through a 75-mil section of DNA contained within a Teflon sample holder. Measurements of the dry sodium herring DNA sample were also taken over a much broader frequency range utilizing a Bell Labs T-Ray Source (Smith et al. IEEE J. Quantum Elec. 1988 24:255; Nuss, M. C. IEEE Circuits Devices March, 1996 25). While unconfirmed by any additional measurements, power-absorption results, for transmission through four different locations of the sample layer in the frequency of 100-400 GHz, were disclosed to result in relatively well-defined peaks resolved at 160, 180, 230, 260, 30 290, 330 and 390 GHz. Based upon these preliminary experiments, it is speculated that microwave absorptions techniques may be useful in detecting DNA-based microorganisms. Further, the feasibility and advantages of using a particular set of modes of the DNA polymers as identification resonances are discussed. However, dry-packing of samples into the waveguide sections as described can lead to inhomogeneity of the sample or possible influence by waveguide eigen-modes. Alternatively, measurement of the samples as thick films can lead to standing waves that tend to mask the signal being detected.
In the present invention a method for inducing and detecting lesion-induced resonance phenomena in thin films of a few micrometers or other samples of DNA molecules via an apparatus comprising an electronically tunable source of electromagnetic radiation capable of generating a broad range of frequencies in the millimeter and submillimeter range wave spectral region, a cavity or sample holder containing the DNA sample and a detector capable of monitoring and recording the radiant power transmitted through the sample as a function of frequency is provided.
An object of the present invention is to provide a method of identifying an unknown DNA molecule in a sample which comprises transmitting through a sample of unknown DNA electromagnetic radiation in a selected range of frequencies in the millimeter or submillimeter wave range; detecting the radiation transmitted through the sample over the selected range; generating an absorbance spectrum which correlates with the detected radiation; and comparing the generated absorbance spectrum with absorbance spectra generated for known DNA samples so that the unknown DNA molecule is identified.
Another object of the present invention is to provide a method of detecting a mutated DNA molecule from a selected species which comprises transmitting through a sample of DNA electromagnetic radiation in a selected range of frequencies in the millimeter or submillimeter wave range; detecting the radiation transmitted through the sample over the selected range; generating an absorbance spectrum which correlates with the detected radiation; and comparing the generated absorbance spectrum with absorbance spectra generated for nonmutated DNA of the same species wherein differences in the absorbance spectra are indicative of a mutation.
Another object of the present invention is to provide a method of identifying agents which mutate DNA which comprises transmitting through a first sample of DNA electromagnetic radiation in a selected range of frequencies in the millimeter or submillimeter wave range; detecting the radiation transmitted through the first sample of DNA over the selected range; generating an absorbance spectrum which correlates with the detected radiation; contacting the first sample of DNA sample with an agent suspected of mutating DNA; transmitting through the DNA sample contacted with the agent electromagnetic radiation in a same selected range of frequencies in the millimeter or submillimeter wave range as the first sample of DNA; detecting the radiation transmitted through the DNA sample contacted with the agent over the selected range; generating an absorbance spectrum which correlates with the detected radiation for the DNA sample contacted with the agent; and comparing the absorbance spectrum of the first DNA sample to the absorbance spectrum of the DNA sample contacted with the agent wherein differences in the absorbance spectra are indicative of the agent being mutagenic.
Yet another object of the present invention is to provide an apparatus for identifying a DNA molecule which comprises a spectrometer composed of a broadband millimeter or submillimeter wave signal source and a detector, a data bank with all spectra of defect-related DNA local modes for a class of substances, and a means for comparing spectra obtained with spectra in the data bank.